Concentricolide and its derivatives, process for preparing them, pharmaceutical composition comprising the same and its use

ABSTRACT

The present invention relates to concentricolide and its derivatives, a method for the preparation of the compound and its derivatives, a pharmaceutical composition containing concentricolide and its derivatives, and use of the compound and its derivatives for the treatment and prevention of infection caused by human immunodeficiency virus (HIV).

TECHNICAL FIELD

The invention relates to concentricolide and its derivatives, a processfor preparing the compound and its derivatives, a pharmaceuticalcomposition comprising concentricolide and its derivatives, and use ofthe compound and its derivatives for the treatment and prevention of theinfection caused by human immunodeficiency virus (HIV).

BACKGROUND ART

Fungi of ascomycete genus Daldinia and other Xylariaceae are rich inunique secondary metabolites. European and American Daldinia sp. havebeen studied by Allport and Bu'Lock (D. C. Allport, J. D. Bulock, J.Chem. Soc., 1958, 4090; D. C. Allport, J. D. Bulock, J. Chem. Soc.,1960, 654.) and Anke et al., (H. Anke, M. Stadler, A. Mayer, O. Sterner,Can. J. Bot., 1995, 73, 802), respectively, thereby resulted in theidentification of characteristic metabolites in their stromata andcultures. Recently, two Japanese Daldinia sp. have been investigatedintensively by Hashimoto and Asakawa (M. Stadler, H. Wollweber, A.Muhlbauer, T. Henkel, Y. Asakawa, T. Hashimoto, Y. M. Ju, J. D. Rogers,H. G. Wetzstein, H. W. Tichy, Mycotaxon 2001, 77, 379; M. S. Buchanan,T. Hashimoto, S. Takaoka, et al., Phytochemstry 1996, 42, 173; D. N.Quang, T. Hashimoto, M. Tanaka, M. Baumgartner, M. Stadler, Y. Asakawa.Phytochemstry 2002, 61, 345; M. Buchanan, T. Hashimoto, and Y. Asakawa,Phytochemstry 1995, 40, 135; M. S. Buchanan, T. Hashimoto, and YAsakawa, Phytochemstry 1996, 41, 821.5-9). More than 20 new metaboliteshave been discovered, and some of these compounds exhibit a wide rangeof biological activities including antimicrobial and nematicidalactivities.

SUMMARY OF THE INVENTION

The inventor has discovered a novel compound having benzonfuran lactoneas backbone from Chinese Daldinia concentrica collected at Lijiang ofYunnan, which compound is named concentricolide A.

The concentricolide exhibits good antifusion biological activity. It canprevent HIV to enter into normal cells, inhibit the reproduction of HIVand delay the destruction of human immune system. Accordingly, theconcentricolide can be used for prevention and/treatment of HIVinfection.

The invention at first aspect relates to compounds of the generalformula (I) or derivatives thereof

In which

-   R1 represents C₁-C₄ alkyl;-   R2 represents H, halogen, —OH, NRR′ or —NO₂, wherein R and R′    represent H or C₁-C₆ alkyl;-   R3 represents H, halogen, —OH, NRR′ or —NO₂, wherein R and R′    represent H or C₁-C₆ alkyl;-   R4 represents H, halogen or —NO₂.

The invention at another aspect relates to a pharmaceutical compositioncomprising, as active ingredients, the compounds of the general formula(I) or derivatives thereof and pharmaceutically acceptable carriers orexcipients.

The invention also relates to a process for preparing the compounds ofthe general formula (I) or its derivatives, comprising:

-   a) extracting the fruiting body of Daldinia concentrica from Yunnan    or its fermented liquid with an organic solvent;-   b) separating the extract by silica gel column chromatography to    give the compound of the formula (II):-   c) subjecting the compound of the formula (II) to bromination,    nitration or alkylation to give the compound of the formula (I).

The invention also relates to the use of the compounds of formula (I)for the manufacture of a medicament for the treatment/prevention of theconditions or diseases associated with HIV infection.

The invention further relates to a method for the prevention /treatmentof conditions or diseases associated with HIV infection, comprisingadministrating the compound of the formula (I) or its derivatives to apatient infected by HIV.

The invention further relates to an extract of Daldinia concentrica fromYunnan, characterized in that the extract contains a compound of formula(II)

The invention further relates to a pharmaceutical compositioncomprising, as active ingredients, an extract of Daldinia concentricaand pharmaceutically acceptable carriers or excipients, in which theextract of Daldinia concentrica contains concentricolide of the formula(II).

The invention further relates to the use of the extract of Daldiniaconcentrica for the preparation of a product for theprevention/treatment of conditions or diseases associated with HIVinfection.

According to the invention, the compound of formula (I) or itsderivatives are selected preferably from the compounds represented bythe following formulae:

more preferably from the compound of formula (II).

According to the invention, the compound of formula (I) or itsderivatives comprise optical stereoisomers thereof, preferably astereoisomer represented by the formula (II).

According to the invention, the term “derivatives of a compound offormula (I)” means pharmaceutically acceptable salts, stereoisomers,hydrates or solvates of the compound of formula (I).

According to the invention, the compounds concerned in this inventionform salts with a variety of organic and inorganic acids, includingphysiologically acceptable salts which are commonly used inpharmaceutical chemistry. Such salts are also included in thisinvention. Examples of typical inorganic acids used to form such saltsinclude hydrochloric acid, nitric acid, sulfuric acid, phosphoric acidand the like. Examples of typical organic acids include: mono- andbi-aliphatic acids, phenyl substituted alkanoic acids, hydroxyalkanoicacids and hydroxyalkandioic acids, aromatic acids, aliphatic andaromatic sulfonic acids and so on. The pharmaceutically acceptable saltsinclude acetates, phenylacetates, trifluoroacetates, acrylates,ascorbates, benoates, chlorobenzoates, ldinitrobenoates,hydroxybenzoates, methoxybenzoates, methylbenzoates, O-acetoxybenzoates,naphthalene-2-benzoates, bromides, isobytyrates, phenylbutyrates,β-hydroxybutyrates, butyne-1,4-dioates, hexyne-1,4-dioates, caprates,caprylates, chlorides, cinnamates, citrates, formates, fumarates,glycollates, heptanoates, hippurates, lactates, malates, maleates,hydroxymaleates, malonates, mandelates, mesylates, nicotinates,isonicotinates, nitrates, oxalates, phthalates, teraphthalates,phosphates, monohydrogenphosphates, dihydrogenphosphates,metaphosphates, pyrophosphates, propiolates, propionates,phenylpropionates, salicylates, sebacates, succinates, suberates,sulfates, bisulfates, pyrosulfates, sulfites, bisulfites, sulfonates,benzene-sulfonates, p-bromophenylsulfonates, chlorobenzenesulfonates,ethanesulfonates, 2-hydroxyethanesulfonates, methanesulfonates,naphthalene-1-sulfonates, naphthalene-2-sulfonates, p-toluenesulfonates,xylenesulfonates, tartarates and the like. The preferred salt ishydrochlorides.

According to the invention, the compound of the formula (II) is preparedfrom fungus material of Daldinia sp. The comminuted fungus material ofDaldinia sp. is extracted with an organic solvent (e.g. methanol,ethanol, propanol, isopropanol, acetone, ethyl acetate, chloroform,diethyl ether, t-butyl methyl ether, tetrahydrofuran, acetonitrile), theextract is concentrated and adsorbed on a solid carrier. The solidcarrier includes ground natural ores (e.g. kaolin, clay, talc, chalk,quartz, montmorillonite) and ground synthetic minerals (e.g. silica,alumina, silicates) or absorber resins (e.g. phenol-formaldehyde resinsor polyamide resins). The absorbed extract is subjected to gradientelution using mixed solvents of increasing polarity, the eluent isconcentrated and further purified on silica gel column.

Alternatively, the compound of formula (II) is prepared by extractingthe fermented liquid cultures of Daldinia concentrica with an organicsolvent (e.g. ethyl acetate, diethyl ether, chloroform, methylenechloride, butanol), concentrating the extract and absorbing on a solidcarrier including ground natural minerals (e.g. kaolin, clay, talc,chalk, quartz, montmorillonite) and ground synthetic minerals (e.g.silica, alumina, silicates) or absorber resins (e.g. phenol-formaldehyderesins or polyamide resins), subjecting the absorbed extract to gradientelution, concentrating the eluent and purifying on silica gel column.

According to the invention, the compounds of the formula (I) or (II) aresuitable for the treatment of acquired immunodeficiency syndrome (AIDS)caused by the infection of human immunodeficiency virus (HIV). Thecompounds can be isolated directly from fungi or be obtained by fullsynthesis or semi-synthesis. All the compounds according to theinvention are used separately or in the form of pharmaceuticalcomposition when they are used for treating the infection caused byhuman immunodeficiency virus (HIV). The invention also concerns to theuse of extracts of Daldinia sp. for the treatment of the infectioncaused by human immunodeficiency virus (HIV). The extracts comprise thecompound of formula (II) and can be used in combination with othercompounds or formulations which are suitable for the treatment of theinfection caused by HIV.

Further, the extracts of Daldinia sp. can be obtained by conventionalextraction steps using above organic solvents. The extracts of variouspurity grades can be used. The content of the compound of the formula(II) in the extract is in the range of 0.01 to 2% be weight, preferablyin the range of 0.1 to 1% by weight.

The invention also relates to the use of fungi of Daldinia sp. for thetreatment of the infection caused by human immunodeficiency virus (HIV).The fungi is preferably in. the form of comminuted fruiting bodies orstromata and cultures. The comminuted fungus can be used together withother compounds or formulations for the treatment of the infectioncaused by HIV.

One or more compounds of formula (I) or (II) are compressed togetherwith excipients, diluents or carriers into tables or other oral dosageforms, or formulated as pharmaceutical dosage forms for intramuscular orintravenous injection, or as sustained release dosage forms and thelike.

Pharmaceutical formulations are prepared by procedures known in the art.In particular, the compounds according to the invention can beformulated with common excipients, diluents or carriers, and formed intotablets, capsules, suspensions, powders and the like. Examples ofexcipients, diluents and carriers include fillers and extenders (such asstarch, sugars, mannitol, and silicic derivatives), binding agents (suchas carboxymethyl cellulose, other cellulose derivatives, alginates,gelatin, and polyvinyl pyrrolidone), moisturizing agents (such asglycerol), slow release agents (such as paraffin), resorptionaccelerators (such as quaternary ammonium salts), surfactants (such ascetyl alcohol, glycerol monostearate), adsorptive carriers (such askaolin and bentonite) and lubricants (such as talc, calcium stearate,magnesium stearate, and solid polyethyl glycols).

The compounds according to the invention can be formulated as non-oraldosage forms, such as the dosage forms for intramuscular, subcutaneousor intravenous injections, or as sustained release dosage forms and thelike. The formulations can be so constituted that they release theactive ingredient in a particular part of the intestinal tract over aperiod of time. The coatings, envelopes, and protective matrices may bemade from polymeric materials or waxes.

The particular dosage of the compound of formula (I) or (II) for thetreatment of the infection caused by the human immunodeficiency virus(HIV) is dependent upon many factors, such as the severity of diseases,body weight, age, sex, administration path, and specific diagnosis ofclinician. Generally, accepted and effective daily doses are from 0.1 to1000 mg/person/day, preferably from 50 to 200 mg/person/day. Suchdosages are administered once to three times each day.

DESCRIPTION OF THE DRAWINGS

FIG. 1 The cytotoxicity of concentricolide A or II on C8166 cells (CC₅₀is 76.66 μg/ml);

FIG. 2 The cytotoxicity of AZT on C8166 cells (CC₅₀is 258.88 μg/ml);

FIG. 3 The blockage of concentricolide A or II on binding and fusionbetween HIV-1 and cells (EC₅₀ is 0.83 μg/ml);

FIG. 4 The blockage of T20 on fusion of HIV-1 infected cells (EC₅₀ is6.02 ng/ml);

FIG. 5 Inhibition of concentricolide A or II on cytopathic effect (CPE)of HIV-1 (EC₅₀ is 0.31 μg/ml);

FIG. 6 Inhibition of AZT on cytopathic effect (CPE) of HIV-1 (EC₅₀ is0.092 μg/ml)

FIG. 7 Lattice diffraction pattern of concentricolide A according to theinvention.

EMBODIMENTS

Following examples are used to further describe the invention, but donot limit the invention at any way.

EXAMPLE 1

Preparation of Concentricolide A

Dried and comminuted fruiting bodies (750 g) of Daldinia concentricawere extracted for three times with CHCl₃—MeOH (1:1, vol/vol) and MeOHat room temperature, respectively. The organic phase was combined andevaporated in vacuo to give a deep brown and cream crude extract (60 g).

The extract was mixed with 80-100 mesh silica gel and submitted tosilica gel chromatography eluting successively with CHCl₃/MeOH (100:0,95:5, 90:10, vol/vol) to give 15 fractions. The fraction 8 (eluted withCHCl₃/MeOH, 95:5, 9 g) was subjected to silica gel chromatographyeluting successively with petroleum ether/acetone (99:1, 95:5, 90:10,80:20, vol/vol). From the fraction eluted with 99:1 petroleumether/acetone was directly obtained 120 mg concentricolide A.

Alternatively, the liquid culture (80 L) of Daldinia concentrica wasextracted for five times with EtOAc at room temperature. The combinedorganic phase was evaporated in vacuo to give a deep brown and creamcrude extract (24 g). The crude extract was mixed with 80-100 meshsilica gel and submitted to silica gel chromatography eluting withCHCl_(3/)MeOH (100:0, 95:5, 90:10, vol/vol) to give 15 fractions. Thefraction 8 (obtained by eluting with CHCl₃/MeOH, 95:5, 1.7 g) wassubjected to gel chromatography eluting with petroleum ether/acetone(99:1, 95:5, 90:10, 80:20, vol/vol). From the fraction eluted with 99:1petroleum ether/acetone was directly obtained 173 mg concentricolide A.

Concentricolide A has the following physico-chemical and wave spectrumdate: pale yellow needles, m.p. 89˜90° C. (petroleum ether-acetone);[α]_(D) ^(21.9)=−59.23°(c 0.48, MeOH); EI-MS m/z (rel. int.): 202[M]⁺(20), 173 (100), 145 (48);HR-TOF-MS m/z: 225.0526 ([M+Na]⁺, 225.0527calcd. for C₁₂H₁₀O₃Na); UV(MeOH)λ_(max) nm: 226 (log ε 4.08), 256.5(3.88), 297 (3.63); IR ν^(KBr) _(max)cm⁻¹: 1757, 1641, 1534, 1437.

¹H- and ¹³C-NMR data of concentricolide (500 MHz, CD₃OD, δ in ppm, J inHz) HMBC δ (C)(DEPT) Δ (H) ¹H-¹HCOSY (selected) CH (2) 146.4(CH)7.74(1H, d, H-3 H-3 J=2.2) CH (3) 106.6(CH) 6.86(1H, d, H-2 H-2, 4J=2.2) C (3A) 128.9(C) H-2, 5 CH (4) 127.8(CH) 7.86(1H, d, H-5 H-3J=8.3) CH (5) 116.0(CH) 7.24(1H, d, H-4 J=8.3) C (5A) 147.8(C) H-4, 9 CH(6)  82.9(CH) 5.52(1H, dd, H-9a H-5, 9, 10 J=7.1, 4.1) C (8B) 110.9(C)H-5, 6 C (8B) 149.6(C) H-2, 3, 4 1.81(1H, m) H-6, 9b, 10 H-6, 10 CH₂ (9) 27.8(CH₂) 2.13(1H, m) H-9a, 10 CH₃ (10)  8.7(CH₃) 0.95(3H, t, H-9a, 9bH-6, 9 J=7.2) C (8) 168.9(C) H-6X-Ray diffraction Analysis Results:

C₁₂H₁₀O₃, M 202, triclinic, space group P1; a=7.728(1), b=8.289(1),c=9.043(1) Å; α=106.450(5)°, β=96.321(6)°, γ=108.946(6)°; V=512.36(3)Å³, Z=2. Final reliable factor R_(f)=0.073 and R_(w)=0.066 (w=1/σ |F|²).A total of 1369 reflections were recorded in the ω scanning mode with aMAC-DIP-2030K diffractometer with graphite-monochromated MoKα scanningradiation. The structure was solved by the direct method SHELXS86. Itslattice diffraction pattern was shown in FIG. 7.

EXAMPLE 2 The preparation of 2,3-dibromo-2,3-dihydroconcentricolide (B)from concentricolide A

60 mg of concentricolide (A, 0.30 mmol) was dissolved in 2 ml CHCl₃, 65mg bromine (0.41 mmol) was dissolved in 1 ml CHCl₃. The bromine solutionwas added dropwise to the solution of A and stirred at room temperaturefor 120 hours. The product was concentrated to give pale yellow needles,which was recrystallized to obtain 50.8 mg of B with a yield of 47.2%.The product showed two peaks on HPLC which are identified to be a pairof optical isomers (B₁ and B₂).

Pale yellow needle compound B was produced by addition reaction ofconcentricolide A and bromine. EI-MS spectrum showed three isotope peaksat 364, 362 and 360 with a relative intense of 1:2:1, indicating thatthe molecular contains two atoms. By comparison with concentricolide A,there is just the difference of molecular weight of two bromine atoms,further proving the existence of two bromine atoms. In ¹H-NMR spectrum,two olefinic hydrogen coupled each other [δ 7.79 (1H, d, J=7.7), 7.19(1H, d, J=7.7)] was observed. Based on coupling constant J=7.7, it isinferred to be hydrogen on the phenyl ring, suggesting the bromineaddition reaction occurred at the position 2 and 3. In HMBC, δ_(H) 7.79was related to C-3, 5A and 8A, and δ_(H) 7.19 was related to C-3A, 4, 5Aand 8A, which further proved that the addition reaction occurred atfuran ring. Therefore, the structure of B was determined to be2,3-dibromine omo-2,3-hydroconcentricolide. HPLC of B showed that thereare two peaks, because bromine addition reaction was bromine onium ionmechanism, two bromine atoms took contrary directions to attack theolefinic carbon. The product of the addition reaction should be a pairof optical isomers, that is, B was indeed a mixture, in which thestructures of the two compounds were(2S,3S)-2,3-dibromo-2,3-hydroconcentricolide (B₁) and(2R,3R)-2,3-dibromo-2,3-hydroconcentricolide (B₂), respectively.

2,3-Dibromo-2,3-dihydroconcentricolide (B₁ and B₂). Pale yellow needles(petroleum ether/acetone)

EI-MS m/z (rel. int.): 364 [M₁]⁺ (0.5), 362[M₂]⁺ (1.0), 360[M₃]⁺ (0.5),284(7), 282 (13), 280 (31), 278 (7), 202 (20), 173 (100), 145 (50), 117(7), 89 (12);

¹H-NMR (500 MHz, CDCl₃): δ7.79 (1H, d, J=7.7), 7.19 (1H, d, J=7.7), 7.02(1H, d, J=3.8), 5.79 (1H, d, J=4.2), 5.46 (1H, m), 2.14 (1H, m), 1.86(1H, m), 1.06 (3H, t, J=7.4);

¹³C-NMR (100 MHz, CDCl₃): δ 166.6, 166.6, 153.9, 153.9, 153.8, 153.8,131.6, 131.5, 128.1, 128.0, 117.1, 117.1, 112.2, 112.0, 90.1, 90.0,82.8, 82.8, 50.9, 50.8, 27.8, 27.7, 9.1, 9.0.

EXAMPLE 3 The preparation of 3-bromoconcentricolide (C) from2,3-dibromo-2,3-dihydroconcentricolide (B)

32 mg B (0.09 mmol) was dissolved in 2 ml methanol, to the solution wasadded 1 ml saturated methanol solution of potassium hydroxide. Themixture was stirred at room temperature for 8 hr. The reaction solutionwas neutralized to pH 7.0 with 1% hydrochloric acid in methanol anddiluted with a small amount of water and extracted with chloroform. Theextract was dried to obtain a crude product. The crude product wasrecrystallized to give 11.8 mg pale yellow needles C with a yield of47.5%.

Pale yellow needles compound C was obtained as an elimination reactionproduct of B. EI-MS spectrum showed two molecular ion peaks at 282 and280 with a relative intense of 1:1, indicating the existence of onebromine atom. Three olefinic hydrogen signals [δ 7.84 (1H, d, J=8.0),7.80 (1H, s), 7.34 (1H, d, J=8.0)] were observed in ¹H-NMR. Comparingthe signals with those of A [δ 7.74 (1H, d, J=2.2, H-2), 6.86 (1H, d,J=2.2, H-3), 7.86 (1H, dd, J=8.3, 3.6, H-4), 7.24 (1H, d, J=8.3, H-5)],the eliminated was H-3. HMBC showed that there were relevant peaks ofδ_(H) 7.80(1H, s) and C-3, 3A, 8B, further proving that the structure ofthe compound was 3-bromoconcentricolide.

3-Bromoconcentricolide (C). Pale yellow needles(petroleumether/acetone); m.p. 88˜89.5° C.; EI-MS m/z (rel. int.): 282 [M₁]⁺ (20),280 [M₂]⁺ (21), 253 [M₁−C₂H₅]⁺ (80), 251 [M₂−C₂H₅]⁺ (100), 225 (52), 223(68); ¹H-NMR (400 MHz, CDCl₃): δ7.84 (1H, d, J=8.0), 7.80 (1H, s), 7.34(1H, d, J=8.0), 5.56 (1H, m), 2.18 (1H, m), 1.84 (1H, m), 0.99 (3H, t,J=7.4); ¹³C-NMR (100 MHz, CDCl₃): δ 167.2, 149.5, 149.2, 144.1, 128.8,126.5, 116.8, 111.6, 98.3, 82.9, 27.8, 8.7; 2D-NMR.

EXAMPLE 4 The preparation of 2,5-dinitroconcentricolide (D) fromconcentricolide A

50 mg A (0.25 mmol) was dissolved in 1 ml CHCl₃, to the solution wasadded 1.5 ml thick sulphuric acid at cooling condition and addeddropwise 1 ml thick nitric acid with stirring. The mixture was stirredat room temperature for 30 minutes and then poured onto smashed ice.Remained acid was removed by using sodium carbonate. The mixture wasdiluted with a small amount of water and extracted with chloroform. Theextract was dried to obtain a crude product, which was recrystallized togive 31.8 mg pale yellow needles D, with a yield of 44%.

2,5-Dinitroconcentricolide (D). Pale yellow needles (petroleumether/acetone); m.p. 203˜205° C.; EI-MS m/z (rel. int.): 292 [M]⁺ (25),263 [M−C₂H₅]⁺ (100); HR-TOF-MS m/z: C₁₂H₈N₂O₇ ([M+Na]⁻ 315.0220, cal.for C₁₂H₈N₂O₇Na 315.0229); ¹H-NMR (400 MHz, CDCl₃): δ8.98 (1H, s), 7.96(1H, s), 6.13 (1H, m), 2.29 (1H, m), 1.77 (1H, m), 0.91 (3H, t, J=7.4);¹³C-NMR(100 MHz, CDCl₃): δ 163.9, 155.3, 149.7, 147.1, 140.9, 128.2,126.8, 114.6, 106.9, 84.1, 26.3, 9.1.

In the formulations, “active ingredient” means a compound of formula (I)or (II).

EXAMPLE 5

Gelatin Capsules

Hard gelatin capsules were prepared using the following methods:Composition Quantity (mg/capsule) Active ingredient 0.1-1000  Starch0-650 flowable starch 0-650 Silicone fluid (350 centistokes) 0-15 

The ingredients were blended, passed through a sieve, and filled intohard gelatin capsules.

EXAMPLE 6

Concentricolide Capsule Composition Quantity (mg/capsule)Concentricolide 100 Starch 100 flowable Starch 397 Silicone fluid (350centistokes) 3

EXAMPLE 7

Tablets Composition Quantity (mg/capsule) Active ingredient 0.1-1000 Microcrystalline cellulose, 0-650 Silica 0-650 Stearate acid 0-15 

The above components were blended and compressed into tablets.

Tablets each containing 0.1-1000 mg of active ingredient were made up asfollows:

EXAMPLE 8

Tablets Composition Quantity (mg/tablet) Active ingredient 0.1-1000Starch 45 Microcrystalline cellulose 35 Polyvinylpyrrolidone 4 (10%aqueous solution) Carboxymethylcellulose sodium 4.5 Magnesium stearate0.5 Talc 1

The active ingredient, starch, and cellulose were passed through a sieveand mixed. To the mixture was added polyvinylpyrrolidone to form asolution. The solution was passed through a sieve to producepharmaceutical granules. The granules were dried at 50-60° C. and passedthrough a sieve. The carboxymethylcellulose sodium and magnesiumstearate which passed through a sieve were mixed and compressed intotablets.

Suspensions each containing 0.1-1000 mg of effective ingredient per 5 mldose were made as follows:

EXAMPLE 9

Suspensions Composition Quantity (mg/5 ml) Active ingredient 0.1-1000Carboxymethylcellulose sodium 50 Syrup 1.25 mg Benzoic acid solution 0.1 ml Flavoring agent q.v. Colorant q.v. purified water balanced up to5 ml

The effective ingredient was passed through a sieve and mixed withcarboxymethylcellulose sodium and syrup to form a uniform paste. Thebenzoic acid solution, flavoring agent and colorant were diluted usingwater with stirring and then added the pharmaceutical paste, andbalanced with water to the required volume.

Biological Activity Assay

Materials and Methods

Reagents and Chemicals

MTT (3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) waspurchased from Amresco; SDS (sodium dodecyl sulfate) was purchased fromServa; AZT (3P-azido-3P-deoxythymidine) was purchased from Sigma; DMF(N, N′-Dimethyl formamine ) was purchased from Shanghai ChemicalReagents Company (China).

Culture Medium

Complete RPMI-1640 medium supplemented with 10% fetal calf serum(Gibco), 2 mML-Glutamine, 10 Mm HEPES, 50 μM 2-Mercaptoethanol, 100,000IU Penicillin, 100 μg/ml Streptomyces sulfate

Compounds

The testing compound was concentricolide A. The positive control: AZT,one of reverse transcriptase inhibitors, and T20, one of fusioninhibitors were used.

Cells and Virus

C8166 and HIV-1_(IIIB) chronically infected H9 cells were donated byMedical Research Council (MRC), AIDS Reagent Project, UK. All cells andvirus were stored and resuscitated by common methods.

EXAMPLE 1

The Cytotoxicity of Concentricolide A on C8166 Cells

C8166 was one of the host cells for HIV-1. 100 μl of 4×10⁵/ml cells wereseeded on microtiter plate, 100 μl of various concentrations ofconcentricolide were added and incubated at 37° C. in a humidifiedatmosphere of 5% CO₂ for 72 h. The cellular toxicity was assessed by MTTcalorimetric assay. The plates were read on Bio-Tek ELx 800 ELISA readerat 595/630 nm (OD_(595/630 nm)). 50% cytotoxic concentration (CC₅₀) wascalculated as follows:Cell viability %=(OD _(exp) −OD _(blank))/(OD _(control) −OD_(blank))×100.

The results were shown in FIGS. 1 and 2.

EXAMPLE 2

The Blockage of Concentricolide A on Fusion Between Normal Cells andHIV-1 Infected Cells

When HIV infected cells were co-cultured with normal T lymphocyte cells,the exterior envelope glycoprotein gp120 expressed on infected cells wasbound to the cellular CD4 receptor of the uninfected CD4⁺ cells,following fusion of cells and formation of syncytia. Compounds target tothis site would inhibit the syncytia formation. Thus, this method couldbe used to detect whether the compounds have effect on binding andfusion between virus and host cell. Concentricolide A in example 1 wasdiluted by five fold on 96-well microtiter plate, 3 repeated wells ineach gradient, 100 μl per well. Negative control well free of compoundsand T-20 positive control well were set up. Then, 50 μl of C8166 cellsat logarithmic growth period (6×10⁵/ml) and 50 μl of HIV-1 chronicallyinfected H9 cells (2×10⁵/ml) were added in each well. The plate wascultured in a humidified incubator at 37° C. and 5% CO₂ for 24 h, andthe syncytial formation was observed under microscope to deduce whetherConcentricolide A blocks the fusion process of virus and cell . Theresults were as shown in FIGS. 3 and 4.

EXAMPLE 3

The Cytopathic Effect (CPE) Inhibition Assay of Concentricolide A onHIV-I Infected C8166 Cells

To 100 μl of a culture medium containing corresponding concentration ofconcentricolide A from example 1 were added C8166 cells (4×10⁵/ml) andHIV-1_(IIIB) at a multiplicity of infection (M.O.I) of 0.06. The finalvolume was 200 μl. AZT was used as positive drug control. The plateswere incubated in a humidified incubator at 37° C. and 5% CO₂. After 3days of culture, the cytopathic effect was measured by counting thenumber of syncytia in each well under an inverted microscope. EC₅₀ (50%Effective Concentration) was the compound concentration inhibiting 50%of syncytia formation. The cytopathic effect (CPE) inhibition%=(1-number of syncytia_(exp)/number of syncytia_(control))×100. Theresults were as shown in FIGS. 5 and 6.

CONCLUSION

The data demonstrated that concentricolide exhibits low cytotoxicity andgood activity against HIV-1 in vitro. The selected index (S.I.) is 222.The target site of concentricolide probably is binding and fusionbetween HIV and cell. So, as a small molecule compound that blocks thebinding and fusion between HIV and cell, concentricolide A has greatsignificance.

1. Compounds of the general formula (I) and their derivatives

in which R1 represents C₁-C₄ alkyl; R2 represents H, halogen, —OH, NRR′or —NO₂, wherein R and R′ represent H or C₁-C₆ alkyl; R3 represents H,halogen, —OH, NRR′ or —NO₂, wherein R and R′ represent H or C₁-C₆ alkyl;R4 represents H, halogen or —NO₂.
 2. Compounds according to claim 1,selected from the compounds represented by the following formulae:


3. Compounds according to claim 1 or 2, selected from the compounds offormula (II):


4. An extract comprising the compound of formula (II):


5. A pharmaceutical composition comprising the compounds according toany one of claims 1-3 or their derivatives, or the extract as claimed inclaim 4, and a pharmaceutically acceptable carrier or excipient. 6.Process for preparing the compounds of formula (I), comprising: a)extracting fruit bodies of Daldinia concentrica of Yunnan or theirfermented liquid with an organic solvent; b) isolating the extract froma) by silica gel chromatographic to give the compound of the formula(II):

c) subjecting the compound of the formula (II) to bromination, nitrationand alkylation to give the compound of the formula (I).
 7. A use of thecompound according to any one of claims 1-3 for the preparation of amedicament for the prevention/treatment of the conditions or diseasesassociated with HIV infection.
 8. The use of the extract according toclaims 4 for the preparation of a medicament for theprevention/treatment of the conditions or diseases associated with HIVinfection.
 9. A composition according to claim 5, wherein said compoundis selected from


10. A method for the prevention and/or treatment of conditions ordiseases associated with HIV infection, comprising administrating thecompounds according to any one of claims 1-3 and the extract accordingto claims 4 to a patient infected by HIV.